SCHOOL OF MEDICINE

Department of Medicine

IU Infectious Diseases Laboratory

Detection of High Risk Human Papillomavirus (HPV)
by Nucleic Acid Amplification

 

Roche COBAS 4800

The cobas 4800 HPV assay is based on two major processes; specimen processing to elute HPV and cellular DNA on the x480 instrument and real-time amplification of HPV DNA using real-time PCR on the z480 instrument. Cervical specimens, collected in liquid based cytology medium are digested and lysed under denaturing conditions with the resulting DNA being purified through absorption onto magnetic glass particles. The DNA is eluted from the glass particles and transferred to a microwell plate containing DNA amplification master mix. The plate is sealed and moved to the real-time instrument, where it undergoes PCR amplification. Fluorescently labeled reporter probes for HPV 16, 18 and a panel of high risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) are bound to their complementary DNA sequence and fluoresce at a specific wavelength when cleaved by nuclease activity during each amplification cycle. The fluorescent intensity is measured at the end of each amplification cycle and can be used to determine the presence or absence of HPV DNA in the specimen.  Amplification of the human β-globin gene is included in each reaction as a processing control. The performance characteristics of this assay were established in this laboratory for cervical specimens collected in PreservCyt solution. All other sample types are unacceptable. The limit of detection is 300 and 600 copies per ml for HPV16/18 and 15-2400 copies per ml for the high risk panel.

 

Roche Linear Array HPV Genotyping Test

HPV typing can be performed using the Roche Linear Array HPV Genotyping Test (Roche Diagnostics, Indianapolis, IN).  We have developed protocols to successfully detect HPV from cervical, vaginal, anal and hand swabs.  We also have a protocol to detect HPV from oral rinses. This test provides HPV type specific information for 37 anogenital types1. Briefly, this test uses a multiplex PCR to amplify a portion of the HPV L1 gene and includes primers for the amplification of the GH20/PC04 human β-globin target.  The human gene target is used for the assessment of specimen adequacy meaning that samples that fail to amplify this target are not considered adequate2.   Our experience has been that β-globin positivity from cervical and vaginal swabs has been approximately 96%3.

 

1.  Brown DR, Marcia L. Shew, Brahim Qadadri, Nicole Neptune, Maria Vargas, Wanzhu Tu, Beth E. Juliar, Timothy E. Breen, and J. Dennis Fortenberry A Longitudinal Study of Genital Human Papillomavirus Infection in a Cohort of Closely Followed Adolescent Women. The Journal of Infectious Diseases 2005;191:182-92.

2.  Ermel A, Qadadri B, Morishita A, et al. Human papillomavirus detection and typing in thin prep cervical cytologic specimens comparing the Digene Hybrid Capture II Assay, the Roche Linear Array HPV Genotyping Assay, and the Kurabo GeneSquare Microarray Assay. J Virol Methods 2010;169:154-61.

3.  Ermel AC, Shew ML, Weaver BA, et al. DNA detection and seroprevalence of human papillomavirus in a cohort of adolescent women. Sexually transmitted infections 2014;90:64-9.